Project Summary/Abstract Vision loss associated with corneal clouding affects ~300,000 individuals in the United States. The thickest layer of the cornea is the stroma; a transparent collagen matrix that tends to draw fluid from the aqueous humor. A layer of corneal endothelial cells between the stroma and aqueous humor provides little physical barrier to this fluid movement but actively extrudes osmolytes (bicarbonate lactate), and thus fluid, from the stroma back into the aqueous humor. Endothelial dysfunction allows fluid to accumulate in the stroma, distorting the matrix and causing it to scatter light. Corneal transplant has remained the definitive treatment for all corneal diseases for over a century, but advances our in our understanding of the genetics of disease and mechanisms of endothelial function have paved the way for development of bioengineered corneas and less invasive treatments. However, we still do not have a complete understanding of how the endothelium works. It is only relatively recently that genetic-linkage studies revealed the critical importance of the membrane transport protein SLC4A11 to endothelial health. We have recently established that SLC4A11 is a pH-sensitive H+ conductor. We hypothesize that SLC4A11 acts as a master regulator of endothelial cell pH, sensing and countering pH disturbance in the vicinity of pH-sensitive osmolyte transporters NBCe1-B (Na+/2HCO ? cotransporter) and MCT1 (H+/lactate? 3 cotransporter). In the first part of our proposal we express SLC4A11 in Xenopus oocytes and, using a combination of ion-selective microelectrodes and voltage-clamp circuitry in order to determine the kinetic parameters governing SLC4A11 action that are necessary to implement SLC4A11 in mathematical models of endothelial fluid transport. We also examine the influence of SLC4A11 on NBCe1-B and MCT1 action. In the second part of our proposal we compare the progression of corneal disease signs in Slc4a11-knockout mice and our novel Nbce1b-knockout mouse to study the long-term effects of disturbed endothelial fluid transport. Finally, we investigate a novel therapeutic strategy to de-swell the edematous corneas of Slc4a11-knockout mice using eye drops. The long term goal of our research program is to understand how endothelial cells perform and balance fluid transport with pH homeostasis and to understand the importance of SLC4A11 to corneal health and disease. This project is aligned with the mission of the Corneal Disease Program of the NEI, to apply knowledge from basic science discoveries to the understanding of the physiology of the cornea and to the treatment of corneal disease.